The present invention relates to spectroscopic imaging of heterogeneous light scattering media, and more particularly, but not exclusively, relates to in vivo imaging of biologic tissue by mapping a fluorescence characteristic of the tissue through the detection of light emitted in response to excitation light from a time-varying light source.
The early detection of disease promises a greater efficacy for therapeutic intervention. In recent years, non-invasive techniques have been developed which have improved the ability to provide a reliable and early diagnosis of various afflictions by detecting biochemical changes in the tissue of a patient. For example, Magnetic Resonance Imaging (MRI) has successfully monitored the relaxation of spin states of paramagnetic nuclei in order to provide biomedical imaging and biochemical spectroscopy of tissues. Unfortunately, the complexity and expense of MRI diagnostics limit its availability--especially as a means of routine monitoring for disease.
Another powerful analytical technique with an increasing number of applications in the biological sciences is fluorescence spectroscopy. These applications include biomedical diagnostics, genetic sequencing, and flow cytometry. To date, there are several industrial and academic institutions developing fluorescent and phosphorescent compounds for observing pertinent metabolites and environmental conditions, such as Ca.sup.++, pH, glucose, pO.sub.2, and pCO.sub.2. With the development of dyes and photodynamic fluorescent agents which excite and re-emit in the near-infrared red (NIR) wavelength regime, non-invasive detection of diseased tissues located deep within tissues may also be possible since red excitation and re-emission light can travel significant distances to and from the tissue-air interface (See Wilson et al., Time-Dependent Optical Spectroscopy and Imaging for Biomedical Applications, 80 Proceedings IEEE pp. 918-30 (1992)).
As exemplified by U.S. Pat. Nos. 5,421,337 to Richards-Kortum et al. and 5,452,723 to Wu et al., several investigators have suggested various procedures to differentiate diseased and normal tissues based on fluorescence emissions through non-invasive external measurements or minimally invasive endoscopic measuring techniques. Unfortunately, these procedures generally fail to provide a viable spatial imaging procedure. One reason imaging based on fluorescence has remained elusive is that meaningful relational measurements of fluorescence characteristics from a random, multiply scattering media, such as tissue, are difficult to obtain. For example, fluorescent intensity, which is a function of the fluorescent compound (or fluorophore) concentration or "uptake," is one possible candidate for imaging; however, when this property is used in an optically dense medium, such as a particulate (cell) suspension, powder, or tissue, the local scattering and absorption properties confound measured fluorescent intensities.
Besides intensity, other properties of selected fluorophores such as fluorescent quantum efficiency and lifetime are also sensitive to the local biochemical environment. As used herein, "fluorescent quantum efficiency" means the fractional number of fluorescent photons re-emitted for each excitation photon absorbed or the fraction of decay events which result in emission of a fluorescent photon. "Fluorescent lifetime," as used herein, is defined as the mean survival time of the activated fluorophore or the mean time between the absorption of an excitation photon and re-emission of a fluorescent photon. Like intensity, measurement of these fluorescence characteristics is often limited to well-defined in vitro applications in the research laboratory or in flow cytometry where issues such as scattering, absorption, and changing fluorophore concentrations can be controlled or measured. Moreover, these limitations generally preclude meaningful fluorescence-based imaging of hidden tissue heterogeneities, such as tumors or other diseased tissue regions which cannot be detected by visual inspection.
Thus, a need remains for a technique to non-invasively image multiply scattering tissue based on one or more fluorescence characteristics which does not require extensive information about intrinsic optical properties of the tissue, and takes advantage of the contrast capability offered by fluorescence yield and lifetime characteristics to aide in the identification of tissue heterogeneities. The present invention satisfies this need.